Progress in the mapping of genes responsible for human diseases is now limited by the scarcity of useful restriction fragment length polymorphism (RFLP) DNA markers. The reliability of detecting carriers of deleterious genes is limited by the availability of intragenic and closely linked genetic markers. Single base change mutations and polymorphisms can now be detected only by laborious and expensive methods. Mutations are useful for studies of gene structure and function, and the design of strategies for treatment of genetic diseases, such as hemophilia A. The proposed research is designed to: (A) further develop a new strategy for detecting single bade differences in human genomic DNA, and (B) map and determine the DNA sequence changes of mutations and polymorphisms in the human factor VIII gene. The proposed experiments have three specific aims: Aim 1: Optimize the denaturing gradient gel blot method for finding single bade differences in human genomic DNA. The PI developed this method for detecting and mapping single bade change mutations in genomic DNA of Drosophila melanogaster. In preliminary studies, the PI used the method to detect polymorphisms in human genomic DNA. Aim 2: Identify and map sequence polymorphisms in normal and mutant factor VIII genes. The frequency, distribution, and genetic linkage of factor VIII gene sequence polymorphisms in both normal individuals and hemophilia A patients will be examined. Aim 3: Determine the DNA sequence of factor VIII gene polymorphisms that are (a) found only in mutants and (b) are useful polymorphic markers. The DNA sequence of selected factor VIII gene polymorphisms will be determined. The distribution and genetic diversity of factor VIII mutations will be examined. The methods to be developed in these experiments should be applicable to analysis of any other genetic locus, in any other organism. The factor VIII gene polymorphisms and mutations identified in these experiments will be useful for diagnosis and treatment of hemophilia A.